Insulin Receptor – Agonists and Antagonists Agents
Curator: Larry H Bernstein, MD, FCAP
SerpinB1 Promotes Pancreatic β Cell Proliferation: Implications for Treatment of Diabetes
http://pharmaceuticalintelligence.com/2015/12/20/serpinb1-promotes-pancreatic-%CE%B2-cell-proliferation-implications-for-treatment-of-diabetes/
Peroxisome proliferator-activated receptor (PPAR-gamma) Receptors Activation: PPARγ transrepression for Angiogenesis in Cardiovascular Disease and PPARγ transactivation for Treatment of Diabetes
http://pharmaceuticalintelligence.com/2012/11/13/peroxisome-proliferator-activated-receptor-ppar-gamma-receptors-activation-ppar%CE%B3-transrepression-for-angiogenesis-in-cardiovascular-disease-and-ppar%CE%B3-transactivation-for-treatment-of-dia/
Overview of New Strategy for Treatment of T2DM: SGLT2 Inhibiting Oral Antidiabetic Agents
http://pharmaceuticalintelligence.com/2012/11/22/overview-of-new-strategy-for-treatment-of-t2dm-sglt2-inhibiting-oral-antidiabetic-agents/
Int J Mol Med. 2013 Jun;31(6):1463-70. doi: 10.3892/ijmm.2013.1335. Epub 2013 Apr 5.
Astragalus polysaccharide induces anti-inflammatory effects dependent on AMPK activity in palmitate-treated RAW264.7 cells.
Lu J1, Chen X, Zhang Y, Xu J, Zhang L, Li Z, Liu W, Ouyang J, Han S, He X.
http://www.ncbi.nlm.nih.gov/pubmed/23563695
Fish Shellfish Immunol. 2014 May;38(1):149-57. doi: 10.1016/j.fsi.2014.03.002. Epub 2014 Mar 20.
Astragalus polysaccharides: an effective treatment for diabetes prevention in NOD mice.
(PMID:18924264)
http://europepmc.org/abstract/med/18924264
Biochem Biophys Res Commun. 2010 Jul 23; 398(2):260-5.
doi: 10.1016/j.bbrc.2010.06.070. Epub 2010 Jun 19.
S961, an insulin receptor antagonist causes hyperinsulinemia, insulin-resistance and depletion of energy stores in rats.
Vikram A1, Jena G.
Impairment in the insulin receptor signaling and insulin mediated effects are the key features of type 2 diabetes. Here we report that S961, a peptide insulin receptor antagonist induces hyperglycemia, hyperinsulinemia ( approximately 18-fold), glucose intolerance and impairment in the insulin mediated glucose disposal in the Sprague-Dawley rats. Further, long-term S961 treatment (15day, 10nM/kg/day) depletes energy storage as evident from decrease in the adiposity and hepatic glycogen content. However, peroxysome-proliferator-activated-receptor-gamma (PPARgamma) agonist pioglitazone significantly (P<0.001) restored S961 induced hyperglycemia (196.73+/-16.32 vs. 126.37+/-27.07 mg/dl) and glucose intolerance (approximately 78%). Improvement in the hyperglycemia and glucose intolerance by pioglitazone clearly demonstrates that S961 treated rats can be successfully used to screen the novel therapeutic interventions having potential to improve glucose disposal through receptor independent mechanisms. Further, results of the present study reconfirms and provide direct evidence to the crucial role of insulin receptor signaling in the glucose homeostasis and fuel metabolism.
Biochem Biophys Res Commun. 2008 Nov 14; 376(2):380-3.
doi: 10.1016/j.bbrc.2008.08.151. Epub 2008 Sep 7.
A novel high-affinity peptide antagonist to the insulin receptor.
Schäffer L1, Brand CL, Hansen BF, Ribel U, Shaw AC, Slaaby R, Sturis J.
Author information
In this publication we describe a peptide insulin receptor antagonist, S661, which is a single chain peptide of 43 amino acids. The affinity of S661 for the insulin receptor is comparable to that of insulin and the selectivity for the insulin receptor versus the IGF-1 receptor is higher than that of insulin itself. S661 is also an antagonist of the insulin receptor of other species such as pig and rat, and it also has considerable affinity for hybrid insulin/IGF-1 receptors. S661 completely inhibits insulin action, both in cellular assays and in vivo in rats. A biosynthetic version called S961 which is identical to S661 except for being a C-terminal acid seems to have properties indistinguishable from those of S661. These antagonists provide a useful research tool for unraveling biochemical mechanisms involving the insulin receptor and could form the basis for treatment of hypoglycemic conditions.
Betatrophin: a hormone that controls pancreatic β cell proliferation
Peng Yi,1 Ji-Sun Park,1 and Douglas A. Melton1,†
Cell. 2013 May 9; 153(4): 747–758. doi: 10.1016/j.cell.2013.04.008
See commentary “The p38–PGC-1α–irisin–betatrophin axis” in Adipocyte, volume 3 on page 67.
See commentary “Betatrophin” in Islets, volume 6, e28686.
Replenishing insulin-producing pancreatic β cell mass will benefit both type I and type II diabetics. In adults, pancreatic β cells are generated primarily by self duplication. We report on a novel mouse model of insulin resistance that induces dramatic pancreatic β cell proliferation and β cell mass expansion. Using this model we identify a new hormone, betatrophin, that is primarily expressed in liver and fat. Expression of betatrophin correlates with β cell proliferation in other mouse models of insulin resistance and during gestation. Transient expression of betatrophin in mouse liver significantly and specifically promotes pancreatic β cell proliferation, expands β cell mass, and improves glucose tolerance. Thus, betatrophin treatment could augment or replace insulin injections by increasing the number of endogenous insulin-producing cells in diabetics.
Diabetes results from dysfunctional carbohydrate metabolism that is caused by a relative deficiency of insulin. It has become a major threat to human health, the prevalence of which is estimated to be 2.8% worldwide (171 million affected), and predicted to rise to 4.4% (366 million) by 2030 (Wild et al., 2004). Around 10% of diabetics in the United States are type I, a disease caused by an autoimmune attack on pancreatic β cells and a consequent β cell deficiency. The majority of diabetics are type II, characterized by interrelated metabolic disorders that include decreased β cell function, peripheral insulin resistance, and, eventually, β cell failure and loss or dedifferentiation (Scheen and Lefebvre, 1996; Talchai et al., 2012). While the disease can be treated with anti-diabetic drugs or subcutaneous insulin injection, these treatments do not provide the same degree of glycemic control as functional pancreatic β cells and do not prevent the debilitating consequences of the disease. Treatments that replenish β cell mass in diabetic patients could allow for the long-term restoration of normal glycemic control and thus represent a potentially curative therapy. Despite the fact that the primary causes for type I and type II diabetes differ, all diabetics will benefit from treatments that replenish their β cell mass.
While there is some evidence that mouse β cells can be derived from rare adult progenitors under extreme circumstances (Xu et al., 2008), the vast majority of new β cells are generated by simple self-duplication (Dor et al., 2004; Meier et al., 2008; Teta et al., 2007). After a rapid expansion in embryonic and neonatal stages, β cells replicate at an extremely low rate (less than 0.5% divide per day) in adult rodents (Teta et al., 2005) and humans (Meier et al., 2008). However, pancreatic β cells retain the capacity to elevate their replication rate in response to physiological challenges including gestation (Parsons et al., 1992; Rieck et al., 2009), high blood sugar (Alonso et al., 2007), pancreatic injury (Cano et al., 2008; Nir et al., 2007), and peripheral insulin resistance (Bruning et al., 1997; Kulkarni et al., 2004; Michael et al., 2000; Pick et al., 1998).
The genetic mechanisms controlling β cell proliferation are incompletely understood. The cell cycle regulators cyclin D1/D2 and CDK4 promote β cell proliferation (Georgia and Bhushan, 2004; Kushner et al., 2005; Rane et al., 1999) and cell cycle related transcription factors such as E2F1/2 are essential for pancreatic β cell proliferation (Fajas et al., 2004; Iglesias et al., 2004). On the contrary, cell cycle inhibitors including p15Ink4b, p18Ink4c and p27Kip1 repress β cell replication (Latres et al., 2000; Pei et al., 2004; Uchida et al., 2005). Other genes reported to regulate β cell proliferation include NFAT, Menin, p53, Rb and Irs2 (Crabtree et al., 2003; Harvey et al., 1995; Heit et al., 2006; Kubota et al., 2000; Williams et al., 1994).
In addition to the factors listed above, which are expressed in β cells themselves and act in a cell-autonomous fashion, there are several reports showing that systematic or circulating factors can regulate β cell replication and mass. Glucose itself is a β cell mitogen; infusion of glucose in rodents causes a mild increase in β cell replication (Alonso et al., 2007; Bernard et al., 1998; Bonner-Weir et al., 1989). And glucokinase defects significantly decrease the compensatory proliferation of pancreatic β cells in some contexts (Terauchi et al., 2007). In addition, genetic deletion of glucokinase in β cells can reduce replication rates, whereas pharmacological activation of this enzyme increases replication by 2 fold (Porat et al., 2011). Several hormones, including insulin, placental lactogen and prolactin also play a role in regulating β cell mass (Bernard et al., 1998; Paris et al., 2003; Parsons et al., 1992; Sachdeva and Stoffers, 2009). The incretin hormones glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP) increase insulin secretion and promote β cell replication (reviewed in (Drucker, 2006)). However, from a therapeutic perspective, the problem with manipulating most of the genes and hormones currently known to impact β cell replication is their lack of β cell specificity and/or the fact that the magnitude of their effect on β cell proliferation is quite modest.
Transplantation studies in mice have shown that insulin resistance results in a circulating islet cell growth factor independent of glucose and obesity (Flier et al., 2001). And in a telling demonstration, the liver specific deletion of the insulin receptor results in a dramatic compensatory increase pancreatic β cell replication (Michael et al., 2000). Similarly, overexpression of a constitutively active MEK1 kinase in mouse liver increases the replication rate in pancreatic β cells and improves glucose tolerance in disease models through an innervation-dependent mechanism (Imai et al., 2008). Precisely how the liver signals pancreatic β cells to proliferate is unknown, but recent work by Kulkarni’s group points to the possibility that liver cells secrete a protein that acts directly on islet cells (El Ouaamari et al., 2013; Flier et al., 2001).
In this study we aimed to identify secreted signals that control pancreatic β cell proliferation. As a first step we developed a novel insulin resistance mouse model wherein β cell replication can be rapidly induced at will. We show that administration of an insulin receptor antagonist induces acute peripheral insulin resistance and leads to a dramatic proliferation in pancreatic β cells and subsequent β cell mass expansion. Using this model, we identified a gene encoding a secreted protein that is expressed in liver and fat and whose expression level is elevated upon insulin resistance. We called this gene betatrophin because its overexpression in mouse liver produces a secreted protein that significantly and specifically promotes pancreatic β cell proliferation, β cell mass expansion, and consequently improves glucose tolerance.
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Results
Administration of an insulin receptor antagonist induces insulin resistance and pancreatic β cell proliferation
Previous work showed that when the insulin pathway is blocked in vivo in the liver pancreatic β cell mass expands and there is an increase in insulin secretion as a compensatory response (Bruning et al., 1997;Michael et al., 2000). To investigate the signals that control this type of β cell compensatory growth, we explored a new pharmacological model of severe insulin resistance. S961 is a peptide (43aa) that binds the insulin receptor and antagonizes insulin signaling both in vitro and in vivo in rats (Schaffer et al., 2008). We used osmotic pumps to infuse adult mice with various doses of S961. The data in Figure 1A show that S961 causes hyperglycemia in a dose dependent manner. A high dose of S961 infused for a week makes the mice glucose intolerant (Figure 1B and 1C), consistent with the fact that S961 blocks the insulin receptor. Plasma insulin levels rise at all doses of the insulin antagonist, presumably due to the compensatory effort of pancreatic β cells (Figure 1D).
Figure 1
Administration of the insulin receptor antagonist S961 induces glucose intolerance, hyperglycemia and hyperinsulinemia
To examine whether S961 induces a compensatory β cell proliferation, as seen in other insulin resistance models, the β cell proliferation rate was analyzed by Ki67 and insulin immunofluorescence for all dosage groups following S961 treatment. S961 treatment results in a dramatic increase in β cell proliferation (Figure 2A), which is both immediate and dose dependent (Figure 2B and Figure S1A–E). The effect of S961 on β cell replication rates is strong, but transient: 4 days after osmotic pump removal, β cell replication rates return to normal (Figure S1F). The proliferation in β cells was confirmed by immunostaining for a nuclear β cell marker (Nkx6.1) and a different cell division marker (PCNA, Figure S2A and S2B). Quantitative PCR analysis of cell cycle regulators shows that the expression level of several Cyclins (Cyclin A1, A2, B1, B2, E1 and F), CDKs (CDK1 and CDK2), E2Fs (E2F1 and E2F2) increase, while the expression of cell cycle inhibitors (Cdkn1a, Cdkn1b and Cdkn2b) decreases in pancreatic islets following S961 treatment (Figure S3A). Even a low dose of S961 (5nmol/week), which does not detectably alter blood glucose levels, produces a modest but reproducible increase in β cell replication (~4.3-fold increase, Figure 2B). At the highest dose tested, S961 treatment resulted in a ~12-fold increase in β cell replication (Figure 2B), a rate vastly exceeding any previously reported pharmacological treatment.
Figure 2
Administration of the insulin receptor antagonist S961 induces pancreatic β cell proliferation and β cell mass expansion
The increase in β cell replication rate appears to affect all pancreatic islets equally (Figure S3B) and leads to an increase in total β cell area of approximately 3-fold within 1 week (Figure 2C–E), primarily resulting from an increase in islet size (Figure S3C). Though β cell mass expands after S961 treatment, pancreatic insulin content decreases (Figure 2F) possibly because β cells secrete more of their insulin into circulation as a consequence of insulin resistance. Though treatment of mice with a low dose of S961 (2.5 nMol/week) does not produce a detectable increase in β cell proliferation at day 7, as measured by Ki67 (Figure 2B), their β cell mass is nonetheless about 1.5-fold higher than the control. Quantification of average β cell size shows no significant difference between vehicle and S961 treated animals (Figure S3D). Thus, the increased β cell mass observed at the low dose of S961 (2.5nMol/week) is not likely due to β cell hypertrophy but rather to the result of a transient increase of β cell proliferation prior to day 7 of S961 treatment. The proliferation induced by S961 administration is highly specific to pancreatic β cells. No obvious differences in cell proliferation rates were noticed, between control and S961 treated animals, for other pancreatic cell types, including other endocrine cells, exocrine cells, and duct cells, nor for liver, white fat or brown fat (Figure 2G).
Identification of Betatrophin in S961 treated mouse liver and white fat
To understand how S961 induces β cell proliferation, we first applied it directly to mouse β cells in vitro to see whether this insulin antagonist works in a β cell autonomous manner, but there was no detectable effect (data not shown). Based on this, we hypothesized that S961 acts indirectly on β cells, and analyzed gene expression in tissues involved in metabolic regulation (liver, white fat, skeletal muscle), in addition to pancreatic β cells themselves, to identify potential mediators of the effect. Microarray analysis pointed to one gene, which we call betatrophin (Figure 3A). Betatrophin is upregulated in S961 treated liver (~4 fold) and white fat (~3 fold), but its expression is unchanged in skeletal muscle and pancreatic β cells (Figure 3B) in response to S961.
Figure 3
Identification and expression of betatrophin
Betatrophin encodes a predicted protein of 198 amino acids (the mouse gene was previously annotated as Gm6484 and the protein as EG624219; the human gene is annotated as C19orf80 and the protein Hepatocellular Carcinoma-Associated protein TD26 (Dong et al., 2004)). The gene has 4 exons and lies within the intron of another gene, Dock6, on the opposite strand (Figure S4A). Betatrophin is highly conserved in all mammalian species examined (Figure S4B), but evidently absent in non-mammalian vertebrates and in invertebrates (data not shown).
Betatrophin is enriched in liver and fat tissues and its expression correlates with high pancreatic β cell proliferation rates
Betatrophin mRNA is expressed in mouse liver and fat, with minimal expression in other tissues examined (Figure 3C), consistent with previous reports (Quagliarini, 2012; Ren et al., 2012; Zhang, 2012). In humans, betatrophin is primarily expressed in the liver (Figure 3D) where betatrophin mRNA levels are more than 250 fold higher than that found in other tissues tested. Betatrophin protein can also be detected by western blotting in human liver (Figure 4J).
Figure 4
Betatrophin encodes a secreted protein
To determine whether betatrophin might be involved in regulating β cell replication in other contexts, we examined betatrophin mRNA expression by quantitative PCR in several physiologically relevant animal models of increased β cell replication. Infusion of the insulin receptor antagonist S961, which causes a dramatic pancreatic β cell proliferation, leads to a 6 fold upregulation of betatrophin in liver and 4 fold in white fat (Figure 3E), consistent with the microarray analysis (Figure 3B). In mouse models of type II diabetes, there is increased pancreatic β cell mass (Bock et al., 2003; Gapp et al., 1983; Tomita et al., 1992;Wang and Brubaker, 2002) and betatrophin mRNA is upregulated 3–4 fold in the liver of both ob/ob anddb/db mice (Figure 3F). β cell replication rates also increase during pregnancy (Karnik et al., 2007) and expression of betatrophin mRNA in the liver increases by about 20 fold over the course of gestation (Figure 3G). Finally, specific depletion of β cells with diphtheria toxin leads to increased β cell replication (Nir et al., 2007). This treatment did not stimulate changes in betatrophin mRNA expression in the liver (data not shown). Together, these results indicate that betatrophin expression may contribute to compensatory pancreatic β cell proliferation in response to physiological challenges, but not in a regeneration response after acute injury.
Betatrophin encodes a secreted protein
How might a protein produced in the liver and fat cause pancreatic β cells to divide? Sequence analysis of mouse and human betatrophin shows a predicted secretion signal at the N-terminus and two coiled coil domains (Figure 4A). To demonstrate that betatrophin is indeed a secreted protein, expression plasmids encoding mouse and human betatrophin, fused with a Myc tag at the C-terminus (referred to as mbetatrophin-Myc and hbetatrophin-Myc), were prepared and used to transfect tissue culture cells and to express betatrophin in mouse liver by hydrodynamic tail veil injection (Song et al., 2002; Yant et al., 2000; Zhang et al., 1999). Ectopic gene expression in the cell line Hepa1-6, and in liver cells in vivo, show Myc- tagged betatrophin protein in vesicle-like structures as expected for a secreted protein (mouse Figure 4B, D and human Figure 4C, E). Myc-tagged betatrophin protein is detected in the supernatant of transfected of 293T cells as well as plasma from mice injected with the expression plasmids (mouse Figure 4F, H and human Figure 4G, I). Betatrophin can be detected in human plasma, demonstrating that endogenous betatrophin is a secreted protein in vivo (Figure 4J).
Expression of betatrophin in liver induces dramatic and specific pancreatic β cell proliferation and improves glucose tolerance in mice
To determine whether betatrophin can promote pancreatic β cell proliferation, we used hydrodynamic injection to deliver betatrophin expression constructs to the liver, one of the normal sites of betatrophin expression. Following injection, 5–10% of liver cells expressed betatrophin (or the control protein, GFP,Figure S5) and this expression persisted for at least 8 days (data not shown). Injection of plasmids encoding betatrophin produces a striking increase in β cell replication (Figure 5A). The β cell proliferation rate in betatrophin injected animals averaged 4.6%, 17 fold higher than the control (GFP injected) rate of 0.27% (Figure 5B), with some individual animals achieving replication rates as high as 8.8% (~33 fold increase). The increased proliferation in β cells in betatrophin injected animals was confirmed by immunostaining for the β cell nuclear marker Nkx6.1 and another cell division marker (PCNA, Figure S2C and S2D). Similar to S961 treated mice, quantitative PCR analysis also shows that the expression level of Cyclins (Cyclin A1, A2, B1, B2, D1, D2 and F), CDKs (CDK1 and CDK2), and E2Fs (E2F1 ad E2F2) increase whereas cell cycle inhibitors (Cdkn1a and Cdkn2a) decrease in islets of betatrophin injected mice compared to control injected mice (Figure S3E). The increase in β cell proliferation was observed in all islets examined (Figure S3F). The increased rate of proliferation is so dramatic that one can easily identify islets and β cells at low magnification simply by the immunostaining for replication (Ki67; Figure 5C).
Figure 5
Overexpression of betatrophin in the liver leads to a specific pancreatic β cell proliferation
The high β cell proliferation rate in betatrophin injected mice leads to a significant expansion of β cell numbers and total pancreatic β cell mass (Figure 5D). After 8 days, the total pancreatic β cell area in betatrophin injected mice is 3 fold higher than in control injected mice (Figure 5E). This increase is the result of having more β cells which in turn increases islet size (Figure S3G). The total pancreatic insulin content also increases (~2 fold) in betatrophin injected mice (Figure 5F).
The stimulation in replication caused by betatrophin expression is largely specific for β cells. As shown in Figure 5C and 5G, there is little if any effect on replication in other pancreatic cell types (exocrine, ductal and non-β-cell endocrine cells) or other organs (liver, white fat and brown fat) (Figure 5G).
To evaluate β cell function, we isolated pancreatic islets from control or betatrophin injected mice and performed a glucose-stimulated-insulin-secretion (GSIS) analysis. As shown in Figure S6, the GSIS of pancreatic islets from betatrophin injected mice is indistinguishable from control GFP injected mice, suggesting that the normal function of β cells was maintained after the β cell proliferation in betatrophin injected animals. In addition, a glucose tolerance test was performed in control or betatrophin injected mice. Mice were fasted for 6 hours before glucose injection, and the data show that betatrophin injected mice have a lower fasting glucose level (Figure 6A) and improved glucose tolerance compared to control injected mice (Figure 6A and as shown by Area Under Curve (AUC), Figure 6B). Betatrophin expression also results in a minor increase in fasting plasma insulin levels (Figure 6C), possibly due to the relative short fasting time or an increased glucose sensitivity.
Figure 6
Overexpression of betatrophin in the liver leads to improved β cell function
Because insulin resistance is a potent stimulus known to induce β cell proliferation, it is formally possible that betatrophin may act by first inducing insulin resistance, which in turn leads to compensatory β cell proliferation by some other mechanism. This possibility seems unlikely since the lower fasting glucose in mice over-expressing betatrophin is inconsistent with an insulin resistant phenotype. Nonetheless, to rule out this possibility, we performed an insulin tolerance test, and found no difference between betatrophin and control injected mice, in contrast to S961 administration (10nMol/week) which produces a strong insulin resistance (Figure 6D). These data show that betatrophin promotes β-cells replication without insulin resistance.
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Discussion
The possibility that the liver produces a signal for β cell proliferation has been suggested before, perhaps most convincingly by Kahn’s work on the LIRKO mouse, a liver specific depletion of the insulin receptor that produces β cell hyperplasia (Michael et al., 2000). Here, using a different method, we show that an insulin receptor antagonist (S961) provides a chemical means of achieving this same phenotype. In a dose dependent manner, provision of S961 induces a rapid and significant increase in β cell replication and islet growth.
The S961 insulin resistance model enabled us to identify betatrophin. There are three recent reports where the Gm6484/TD26 gene was identified as a liver and fat enriched gene. Those authors pointed to a possible lipoprotein lipase inhibition activity or an effect on serum triglyceride regulation (Quagliarini, 2012; Ren et al., 2012; Zhang, 2012), but did not report any effects on pancreatic β cell biology, carbohydrate metabolism or diabetes. Our findings on betatrophin suggest that this hormone can regulate metabolism by increasing insulin production via an increase in β cell mass.
The upregulation of betatrophin observed during pregnancy and in the ob/ob and db/db diabetic mouse models, may explain how β cell proliferation and β cell mass is expanded in those instances. In other genetic manipulations that increase β cell replication, such as LIRKO and MEK1 mutations (Imai et al., 2008;Michael et al., 2000), it remains to be determined whether betatrophin is similarly upregulated.
The stimulation of β cell replication we report with S961 and following injection of betatrophin DNA is noteworthy for the rapidity and magnitude of the effect. β cell replication rate is elevated 4 fold during gestation (Karnik et al., 2007), 2–4.5 fold with high glucose infusion (Alonso et al., 2007), 2.6 fold from exendin-4 treatment (Xu et al., 1999), 4 fold in a β cell ablation model (Nir et al., 2007), and 6 fold in LIRKO mice (Okada et al., 2007). S961 treatment can increase β cell replication by 12 fold and providing betatrophin by DNA injection increased replication by an average of 17 fold within a few days making this an exceptionally potent activity. Together these results point to the importance of making recombinant betatrophin protein and testing it directly by injection for effects on β cell mass.
We do not yet know the mechanism of action for betatrophin. It may act directly or indirectly on β cells to control their proliferation. Identification of the betatrophin receptor and/or other possible co-factors will help explain how the liver and fat interact with the pancreas to regulate β cell mass. Nonetheless, identification of betatrophin as a hormone that can exert control on β cell replication and β cell mass opens a new door to possible diabetes therapy.
Agonism and Antagonism at the Insulin Receptor
Louise Knudsen , Bo Falck Hansen, Pia Jensen, Thomas Åskov Pedersen, Kirsten Vestergaard, Lauge Schäffer, Blagoy Blagoev, Martin B. Oleksiewicz, Vladislav V. Kiselyov, Pierre De Meyts
PLOS One Dec 27, 2012 DOI: 10.1371/journal.pone.0051972
Insulin can trigger metabolic as well as mitogenic effects, the latter being pharmaceutically undesirable. An understanding of the structure/function relationships between insulin receptor (IR) binding and mitogenic/metabolic signalling would greatly facilitate the preclinical development of new insulin analogues. The occurrence of ligand agonism and antagonism is well described for G protein-coupled receptors (GPCRs) and other receptors but in general, with the exception of antibodies, not for receptor tyrosine kinases (RTKs). In the case of the IR, no natural ligand or insulin analogue has been shown to exhibit antagonistic properties, with the exception of a crosslinked insulin dimer (B29-B’29). However, synthetic monomeric or dimeric peptides targeting sites 1 or 2 of the IR were shown to be either agonists or antagonists. We found here that the S961 peptide, previously described to be an IR antagonist, exhibited partial agonistic effects in the 1–10 nM range, showing altogether a bell-shaped dose-response curve. Intriguingly, the agonistic effects of S961 were seen only on mitogenic endpoints (3H-thymidine incorporation), and not on metabolic endpoints (14C-glucose incorporation in adipocytes and muscle cells). The agonistic effects of S961 were observed in 3 independent cell lines, with complete concordance between mitogenicity (3H-thymidine incorporation) and phosphorylation of the IR and Akt. Together with the B29-B’29 crosslinked dimer, S961 is a rare example of a mixed agonist/antagonist for the human IR. A plausible mechanistic explanation based on the bivalent crosslinking model of IR activation is proposed.
The insulin receptor (IR) is a member of the receptor tyrosine kinase (RTK) family [1]–[6], which includes the receptors for insulin, insulin-like growth factors (IGFs) and many other growth factors. The RTKs consist of an extracellular portion containing the ligand binding sites, a transmembrane helix, and an intracellular portion with tyrosine kinase activity. Ligand binding triggers activation of the tyrosine kinase activity, involving autophosphorylation of tyrosines around the catalytic site [7]. The extracellular domain of the IR exists under two alternatively spliced forms, IR-A and IR-B, depending on the absence or presence, respectively, of a 12 amino acid segment encoded by exon 11 [3], [4]. The intracellular portion of the IR contains seven tyrosine phosphorylation sites, two in the juxtamembrane domain (JM), Y965 and Y972, three in the tyrosine kinase (TK) domain, Y1158, Y1162, and Y1163, and the last two in the carboxy-terminal tail, Y1328 and Y1334 (IR-B numbering).
The binding of insulin to the IR is described by a curvilinear Scatchard plot, which suggests the existence of high- and low-affinity binding sites and/or negative cooperativity [8]. Furthermore, dissociation of prebound labelled insulin from the IR is accelerated by an excess of non-labelled insulin in comparison to dissociation in buffer alone, a hallmark of negative cooperativity [9]. At supraphysiological concentrations of non-labelled insulin (above 100 nM), the accelerated dissociation of labelled insulin is abolished due to self-antagonism. Models describing these complex binding interactions between insulin and the IR were proposed in 1994 by Schäffer [10] and De Meyts [8]. Both models assume that each IR half contains two binding sites, sites 1 and 2. The insulin molecule crosslinks the two IR halves by binding to site 1 on one α-subunit and site 2 on the other α-subunit, thereby creating a high-affinity interaction, leaving the other two IR sites for interaction with insulin with a lower affinity. In order to explain the acceleration of dissociation of prebound labelled insulin by unlabelled insulin (negative cooperativity), De Meyts [8] proposed that IR sites 1 and 2 are disposed in an antiparallel symmetry, allowing alternative crosslinking of the two pairs of binding sites. In 2006 the crystal structure of the ectodomain dimer of IR was solved [11] and confirmed the antiparallel arrangement of the binding sites. A 5-parameter mathematical model for this complex interaction was recently developed by Kiselyov et al. [12] based on the concept of a harmonic oscillator, which was able to reproduce the essential kinetic features of the ligand-receptor interaction and to provide robust estimates of the parameters (site rate constants and crosslinking constant). Recently, by using the model, the differences in insulin binding kinetics between the two IR isoforms were determined allowing accurate determination of the binding kinetics of the individual sites as well as the apparent affinities [13].
Interestingly, despite the apparent complexity and multi-subsite nature of the binding interaction, all natural ligands of the IR (animal insulins) as well as dozens of chemically modified or genetically engineered insulin analogues over the past four decades were always found to have full agonistic properties with widely divergent potencies in metabolic bioassays like rodent adipocytes lipogenesis (same maximum with dose-response curves shifting left or right). The only exception was a covalent insulin dimer crosslinked between the two B29 lysines, which showed both antagonistic and partial agonistic properties [14]. The mitogenic properties of the IR (e.g. in 3H-thymidine incorporation assays) have not been as thoroughly investigated for possible antagonism, again with the exception of the crosslinked dimer which antagonized mitogenesis [14].
In 2002, peptides binding to the IR binding sites were generated by phage display [15] in order to define the molecular architecture of the receptor and to identify the critical regions (“hotspots”) required for biological activity in a site-directed manner. Two groups of phage-derived peptides were found to bind to or close to the two insulin-binding sites. A third group of phage-derived peptides did not compete for binding to insulin sites 1 and 2, and were therefore named site 3 peptides. Surprisingly, some of the site 1 peptides stimulated glucose uptake in adipocytes with partial or full agonistic activity, even though they were presumably not able to crosslink the IR. In contrast, site 2 and 3 peptides acted as glucose uptake antagonists. In terms of IR phosphorylation, site 1 peptides acted as either agonists or antagonists, whereas site 2 and site 3 peptides acted only as antagonists. Finally, site 1 peptides also bound to the IGF-IR, in contrast to site 2 and 3 peptides, which bound exclusively to the IR [15].
Several combinations of homo-and heterodimers of site 1 and 2 peptides were generated in order to increase the affinity for the IR and to achieve a more insulin-like activation mechanism of the IR [16]. Interestingly, heterodimers of site 1 and 2 peptides acted as either agonists or antagonists, depending on the order of peptide linkage. Heterodimers comprising a site 1 peptide C-terminally linked to the N-terminal end of a site 2 peptide acted as antagonists (these heterodimers are termed site 1–2 peptides). In contrast, heterodimers comprising a site 2 peptide C-terminally linked to the N-terminal end of a site 1 peptide acted as agonists (these heterodimers are termed site 2–1 peptides) [16]. However, Jensen et al. [17] recently found that a site 2–1 peptide named S597 was a full agonist on glycogen synthesis (with a decreased potency), but a weak inducer of cell proliferation in rat L6 myoblast cells overexpressing the human IR-A. Interestingly, the authors found that S597 was able to antagonize the effect of insulin on cell proliferation down to the effect of S597 alone, indicating that S597 is not a full but a partial agonist for mitogenesis [17]. This prompted us to examine more closely the properties of the site 1–2 peptide S961, nearly identical to S661 [18] reported to be a full IR antagonist, and investigate whether it may also have agonistic properties on the IR.
S961 Stimulated a Mitogenic Response in L6-hIR Cells
Usually, in mammalian cells, IGF-I is a stronger mitogen than insulin [20], [21]. However, in L6-hIR cells, insulin and IGF-I had mitogenic potencies (EC50 values) of 0.13 nM and 5.41 nM, respectively (Fig. 1). In this regard, L6-hIR cells are unusually responsive to the mitogenic effect of human insulin. This was in agreement with a previous report [19], supporting that in L6-hIR cells, the mitogenic effect of insulin is primarily mediated by the transfected human IR.
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Figure 1. S961 has antagonist as well as agonist activity on IR-mediated mitogenic effect in L6-hIR cells. A,
“10 nM S961” and “100 nM S961” curves: Cells were pretreated for 2h with 10 nM or 100 nM S961, and stimulated with increasing concentrations of insulin (as indicated on the x-axis) in the continued presence of S961. “HI” curve, insulin stimulation only (without S961). “DMSO” curve, insulin stimulation with equal volume DMSO added instead of S961. B, “10 nM S961” and “100 nM S961” curves: Cells were pretreated for 2h with 10 nM or 100 nM S961, and stimulated with increasing concentrations of IGF-I (as indicated on the x-axis) in the continued presence of S961. “IGF-I” curve, IGF-I stimulation only (without S961). “DMSO” curve, IGF-I stimulation with equal volume DMSO added instead of S961. C, “0.01 nM HI”, “0.025 nM HI” and “0.05 nM HI” curves: Cells were pretreated for 2 h with increasing concentrations of S961 (as indicated on the x-axis), and stimulated with 0.01 nM, 0.025 nM or 0.05 nM HI in the continued presence of S961. “S961 alone” curve, insulin was omitted. D, “1 nM HI”, “10 nM HI” and “100 nM HI” curves: Cells were pretreated for 2 h with increasing concentrations of S961 (as indicated on the x-axis), and stimulated with 1 nM, 10 nM or 100 nM HI in the continued presence of S961. “S961 alone” curve, insulin was omitted.A and B, Graphs are representative for three independent experiments, each experiment comprising triplicate determinations of each ligand concentration. C, The graph is performed in triplicates once. D, The graph is representative for two independent experiments each performed in triplicates. Error bars indicate one standard deviation.
doi:10.1371/journal.pone.0051972.g001
First, because the initial assumption was that S961 is a pure antagonist [18] we performed L6-hIR cell proliferation assays where cells were pre-treated for 2h with 10 nM or 100 nM S961, followed by insulin or IGF-I stimulation in the continued presence of S961. Negative controls consisted of insulin and IGF-I stimulated cells that received an equivalent volume of DMSO instead of S961. At S961 concentrations of 100 nM, the mitogenic potency of human insulin was reduced 100-fold (Fig. 1A), and the mitogenic potency of human IGF-I was reduced 10-fold (Fig. 1B), as shown by the rightward shift of the dose-response curves. In the absence of S961, insulin at below 10 pM and IGF-I at below 1 nM did not stimulate mitogenic responses in L6-hIR cells, as expected (Fig. 1A and 1B). Surprisingly, in the presence of S961 at 10 nM, cell proliferation was observed even at insulin levels below 10 pM and IGF-I levels below 1 nM (Fig. 1A and 1B). Both for insulin in the 0.1 – 10 pM range, and IGF-I in the 0.1 pM – 1 nM range, the increased cell proliferation at 10 nM S961 compared to 100 nM S961 was highly statistically significant (Fig. 1A and 1B, P<0.0005, two-tailed t-test). These results suggested that S961 had not only antagonistic but also agonistic properties.
In order to verify the agonistic effects of S961, we performed a dose-response curve with S961 alone in L6-hIR cells. At concentration of 1 nM, S961 significantly enhanced cell proliferation in comparison to 0.01 nM, (Fig. 1C, P<0.005, two-tailed t-test), whereas the increase in cell proliferation at 10 nM S961 was not statistically significant (Fig. 1C, P = 0.055, two-tailed t-test). At 100 nM S961, the mitogenic effect disappeared (Fig. 1C, “S961 alone” curve). Together, these findings supported that S961 was a mixed agonist/antagonist, with antagonist effects dominant above 10 nM, and agonist activities dominant in the 1–10 nM range, resulting in a bell-shaped curve.
We then examined the effect of low concentrations of insulin on S961-treated cells. The insulin concentrations chosen for this were 0.01 nM, 0.025 nM and 0.05 nM, just at and slightly above the threshold concentration where insulin started to stimulate a mitogenic response in L6-hIR cells (Fig. 1A, “HI” curve). At S961 concentrations of 1 and 10 nM, which corresponded to the maximal agonist activity of S961, the three insulin concentrations did not further increase 3H-thymidine incorporation (Fig. 1C, compare all curves at the 1 and 10 nM x-axis point). In contrast, at S961 concentrations below 1 nM, the low insulin concentrations stimulated an additive mitogenic response (Fig. 1C, compare all curves in the 0.001–0.1 nM x-axis range. P<0.05, two-tailed t-test). This supported that S961 does not exhibit antagonistic activity below 1 nM.
Finally, we examined maximal and supramaximal insulin concentrations corresponding to the maximal mitogenic effect of insulin in S961-pretreated cells (Fig. 1D). This experiment confirmed that above 10 nM, S961 is a strong IR antagonist. Approximately 10-fold molar excess of S961 was needed to neutralize the mitogenic effect of insulin in L6-hIR cells (Fig. 1D).
In summary, all mitogenicity results from L6-hIR cells were concordant, supporting that S961 was a mixed agonist/antagonist, with antagonistic effects dominating above 10 nM and agonistic effects dominating in the 1–10 nM range.
S961 Stimulated a Mitogenic Response in MCF-7 Cells
In order to examine the dose dependant S961 effects on mitogenicity in cancer cells expressing endogenous IR and IGF-IR we performed 3H-thymidine incorporation in MCF-7 cells with S961 and IGF-I. S961 at 1 nM but not at higher concentrations significantly increased cell proliferation in MCF-7 cells (Fig. 2), although to a lesser degree than in L6-hIR cells, showing that the agonistic effect of S961 was not an artefact of the L6-hIR cell system.
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Figure 2. Agonistic (mitogenic) effect of S961 in MCF-7 cells.
Cells were stimulated with increasing concentrations of S961 or IGF-I. The graph is representative for three experiments. The increased response for S961 at 1 nM compared to the response at the three lowest concentrations is statistically significant (P<0.001, two-tailed t-test). Data points represent means of triplicate determinations. Error bars show one standard deviation.
doi:10.1371/journal.pone.0051972.g002
S961 Stimulated IR and Akt Phosphorylation in CHO-hIR Cells
We showed that S661, which has been previously reported to perform in a similar way as S961[18], behaved as an antagonist with respect to IR and AKT phosphorylation (Fig. S1), thus confirming the antagonistic properties of the peptide. S961 concentrations of 1 and 10 nM significantly stimulated tyrosine phosphorylation of the IR (Fig. 3A–E), including the three sites in the tyrosine kinase domain critical for IR activation (Fig. 3B and 3C), i.e. Y1158 and Y1162/1163 in the TK domain, as well as Y1328 and Y1334 in the C-terminal tail end of the IR, and Y972 in the JM domain. Furthermore, S961 concentrations of 1 and 10 nM significantly stimulated Akt phosphorylation at serine 473, known to be critical for the activation of Akt [25](Fig. 4F).
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Figure 3. S961 stimulates IR and Akt phosphorylation in CHO-hIR cells.
Cells were stimulated with increasing concentrations of HI or S961. A–E, IR tyrosine phosphorylation. The 6 tyrosine phosphorylation sites which were examined were Y972 in the juxtamembrane domain, Y1158 and Y1162/1163 in the tyrosine kinase domain, and Y1328 and Y1334 in the C-terminal tail end of the IR. F, Akt phosphorylation. Phosphorylation of Ser437 is known to be required for Akt activation. Panels A–E: the increased tyrosine phosphorylation of the IR was significant (compared to 0.0001 nM, 0.001 nM and 0.01 nM S961, P<0.05*, P<0.01**, P<0.001***, two-tailed t-test). Panel F: the increased serine phosphorylation of Akt was significant (compared to 0.0001 nM, 0.001 nM and 0.01 nM S961, P<0.01**, two-tailed t-test). Data points represent average of three independent experiments, each comprising triplicate determinations. Error bars show one standard deviation.
doi:10.1371/journal.pone.0051972.g003
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Figure 4. S961 did not stimulate glycogen synthesis in differentiated adipocytes or in muscle cells. A
, Differentiated 3T3-L1 adipocytes were stimulated with increasing concentrations of HI, IGF-I or S961. The graph is representative of two independent experiments each comprising duplicate determinations. Error bars show one standard deviation. B, L6-hIR muscle cells were stimulated with increasing concentrations of S961/S661 alone or in combination with 3 nM insulin. The graph is representative of two independent experiments each comprising triplicate determinations. Error bars show one standard deviation.
doi:10.1371/journal.pone.0051972.g004
The S961 dose-response curves for IR and Akt phosphorylation in CHO-hIR cells and the dose-response curves for mitogenicity in L6-hIR and MCF-7 cells coincided perfectly, with maximum at 1 and 10 nM peptide (compare Fig. 1C and 1D with Fig. 2 and Fig. 3A–E).
S961 did not Stimulate Glycogen Synthesis in Differentiated Adipocytes or in Muscle Cells
We investigated if S961 was able to stimulate other biological endpoints than cell proliferation. We therefore performed glycogen synthesis assays with HI, IGF-I and S961 in differentiated 3T3-L1 adipocytes (Fig. 4A) and with S961 alone or in combination with HI in L6-hIR cells (Fig. 4B). As expected, HI and IGF-I were strong and very weak stimulators, respectively, of glycogen synthesis in differentiated 3T3-L1 cells in contrast to S961 which did not induce glycogen synthesis in differentiated adipocytes (Fig. 4A). Similarly, neither S961 nor S661 were able to stimulate glycogen synthesis in L6-hIR cells (Fig. 4B). In addition, both S961 and S661 antagonized the effect of 3 nM insulin with identical potency (Fig. 4B). S661 was included in this experiment to verify peptides similarity.
S961 did not Induce Lipogenesis in Adipocytes
To rule out the possibility that S961 was able to initiate other metabolic pathways than glycogen synthesis, we performed lipogenesis in rat adipocytes. Consistent with the results from glycogen synthesis, S961 and S661, in contrast with insulin, were not able to initiate an agonistic response, but were fully capable of antagonizing the effect of 1 nM insulin (Fig. 5).
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Figure 5. S961 did not stimulate lipogenesis in rat adiopocytes.
Primary rat adipocytes were stimulated with increasing concentrations of S961or S661 alone or in combination with 1 nM insulin. Insulin alone was included as a reference. The graph is representative of two independent experiments each comprising duplicate determinations. Error bars show one standard deviation.
doi:10.1371/journal.pone.0051972.g005
Discussion
Agonism and antagonism at orthosteric or allosteric sites are pharmacological properties of receptors that are well described for the GPCRs [26] and growth hormone/cytokine classes of receptors [27]. Self-antagonism in the latter class of receptors has also been described, resulting in bell-shaped dose-response curves [27], [28]. In the case of RTKs, various strategies to design agonists or antagonists are possible, as described in ref. [29]. Small molecules aimed at inhibiting the TK domain (tyrphostins) have been described for the EGF and other growth factor receptors [30]. Monoclonal antibodies with antagonistic properties have been used successfully to target the ErbB2 receptor, and have made it to the clinic as anti-cancer therapies [31].
In the case of the IR, no natural ligand (various animal insulins) or modified ligand (analogues) has ever been found to be antagonistic in metabolic assays (such as lipogenesis in isolated rodent fat cells) despite the study of dozens of modified insulins. The sigmoid dose-response curves exhibit variable potencies (with leftward or rightward shift relative to insulin) but with the same maximal response. A natural mutant insulin (Leu B24 insulin) was initially claimed to be an antagonist in vitro [32], [33] but was soon demonstrated by others not to be an antagonist either in vitro [34]–[36] or in vivo [37]. A notable exception is a covalent insulin dimer crosslinked between the two B29 lysines, which showed antagonistic and partial agonistic properties in both metabolic and mitogenic assays [14]. The only property of the IR for which antagonism with several insulin analogues has been demonstrated is the negative cooperativity [8]. Dose-response-curves for acceleration of dissociation of pre-bound labelled insulin by unlabelled insulin in an infinite dilution is bell-shaped [8], [9], indicating self-antagonism. Some insulin analogues modified at the C-terminal end of the B-chain (“cooperative site”) [38] or at the N-terminal end of the A-chain (Aladdin H. and De Meyts, P. unpublished data) do not induce the accelerated tracer dissociation and antagonize the accelerating effect of native insulin [8]. These features are readily explainable in the framework of the harmonic oscillator model of the IR [12]. A variety of monoclonal antibodies for the IR and IGF-IR have been shown, depending on their binding epitopes, to be either agonists, neutral or antagonists [39]–[42]. More recently, some monomeric and dimeric peptides targetting IR site 1 and site 2 (described in the introduction) were shown to behave as antagonists of biological effects of insulin in vitro and in vivo [16], [18].
We have investigated here more closely the properties of the site 1–2 dimeric peptide S961, similar to S661 that was previously described as an antagonist [18]. Using three different cell lines (L6-hIR, MCF-7 and CHO-hIR), we showed that S961 is in fact a mixed agonist/antagonist on mitogenic signalling from the IR and that S961 has agonistic effects on IR phosphorylation and Akt phosphorylation endpoints. In all 3 cell lines, S961 exhibited agonistic activity between 1 and 10 nM. The results from all 3 cell culture systems were highly consistent. Thus, the mixed agonist/antagonist properties of S961 were unlikely to be a cell culture artefact. Intriguingly, the agonist activity of S961 was observed only with mitogenicity and IR/Akt phosphorylation endpoints. On the glucose incorporation endpoint in differentiated 3T3-L1 preadipocytes, in L6-hIR cells and in rat adipocytes S961 had no agonistic effects. In addition, we found that S661 behaved in the same manner as S961 with respect to lipogenesis and glycogen synthesis.
Based on the EC50 values of HI and IGF-I, the mitogenic effect of insulin in L6-hIR cells can be reasonably assumed to be mediated by the transfected human IR-A. Additionally, S961 has been reported to be highly IR-specific, with a selectivity for the IR versus the IGF-IR that is higher than that of insulin itself (the IGF-IR affinity of S961 in comparison to HI is 3%, and the IR-A affinity of S961 in comparison to HI is 60% [18]). In addition, a contribution from IR/IGF-IR hybrids [43] is likely since S961, unlike insulin, binds to hybrid receptors with high affinity[18]. In MCF-7 cells, the agonistic effect of S961 is likely induced through IR/IGF-IR hybrids[43]. Indeed, while the cell line we used was shown to contain IR protein by Western blotting[21], we have not been able to demonstrate any high affinity binding of 125I-insulin (Klaproth, B., and Sajid, W., unpublished data), suggesting that most of the IRs are drawn into hybrids which are unresponsive to insulin [43] but bind S961 [18] and IGF-I with high affinity. Also, we showed that the insulin-induced mitogenicity in these cells is not affected by siRNAs against the IR but only by siRNAs against the IGF-IR [44], suggesting that the insulin response is entirely through the IGF-IR. Since S961 binds poorly to the IGF-IR and there are no high-affinity IRs, the response must be through the hybrid receptors for which S961 has a high affinity. Finally, we show that the dose-response curve of S961-induced IR and Akt phosphorylation exactly matched the dose-response of S961-induced mitogenic effect. Therefore, taken together, we believe that our data strongly supported that the mixed agonist/antagonist activity of S961 was exerted through the IR and/or IR/IGF-IR hybrids. A hybrid receptor-mediated response may explain the fact that S961′s agonistic response shows a similar potency in cells that express mostly IRs (L6-hIR cells) or IGF-IRs (MCF-7 cells).
S961 has recently been used in rats as an IR antagonist, to block metabolism as well as mitogenic effects of the IR [45], [46]. We found that in the 1–10 nM range, S961 can in fact act as an agonist of IR-mediated mitogenic responses. Even though we did not find any agonistic effects of S961 on glycogen synthesis in differentiated preadipocytes or in L6-hIR cells as well as on lipogenesis in rat adipocytes, it cannot be ruled out that S961 could have agonistic effects in other cell types. Thus, our findings suggest that when using S961 as an IR antagonist in vitro, S961 concentrations well above 10 nM should be employed.
To our knowledge, together with the B29-B’29 crosslinked dimer, S961 is a rare example of mixed agonism/antagonism at the IR. Another peptide, S597 (a site 1-site 2 peptide), was previously shown to be a full agonist with respect to glycogen synthesis, but a partial agonist on cell proliferation in the presence of HI [17]. The 43 [18] and 31 [17] amino acids long peptides, S961 and S597, have structural similarities since they both consist of a site 1 and site 2 peptide although linked in different orders. None of the peptides show sequence similarity with HI although they were found to bind to the same IR binding sites as HI. The difference between the two peptides could be due to the orientation of the site 1 and site 2 peptides [47].
It is not established how the mixed agonist/antagonist properties of S961 arise. A plausible mechanism can be proposed based on the data presented in our study, and the current model of IR activation [12] which is schematically depicted in Fig. 6A. In this model, the IR molecule has two identical pairs (termed crosslinks) of partial sites (site 1 and site 2) arranged in an anti-parallel way. Insulin can bind first to any of the four available partial sites and then bind to the second site of the same crosslink (see Fig. 6A). It is believed that the crosslinked state of the receptor (with insulin bound to both partial sites) corresponds to the activated state of the receptor [8], [10].
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Figure 6. Current model of IR activation and proposed binding mechanism for S961. A
. Current model of IR activation. The four blue circles represent the receptor binding sites (sites 1 and 2) seen from a top view. Insulin is depicted as a yellow circle. For a detailed explanation of binding sites 1 and 2, see [24]. B. Proposed binding mechanism for S961. The four blue circles represent the receptor binding sites (sites 1 and 2) seen from a top view. For a detailed explanation of binding sites 1 and 2, see [24]. The S961 peptide (Site 1–2 peptide) is shown as two connected yellow circles. At concentrations of 1–10 nM, S961 crosslinks the receptor, leading to agonist activity. At concentrations of above 10 nM, the higher flexibility of S961 in comparison to the insulin molecule allows simultaneous crosslinking of both pairs of binding sites, leading to an inactive conformation and antagonism. The corresponding activation and inactivation sigmoids are also shown. C. Orientation of peptide binding sites. If site 1 is located N-terminally and site 2 C-terminally, a longer distance between the binding sites in S961 in comparison to S661 can be achieved.
doi:10.1371/journal.pone.0051972.g006
The simplest model that can explain mathematically the bell shaped dose response of S961 is a two-site binding model, in which binding to one site activates the receptor and to the second site of lower affinity – inactivates it. Since IR has two identical pairs of partial sites, it is plausible to suggest that binding of the S961 peptide to the first pair of partial sites activates the receptor in a similar way as insulin does (see Fig. 6B). It is known that a second insulin molecule cannot bind simultaneously to the two partial sites of the second pair. However, it is hypothesised that the S961 peptide due to its flexibility can bind simultaneously to the two partial sites, albeit with a lower affinity. The second crosslinking event is postulated to result in the receptor inactivation, which might be a result of formation of a symmetrical “non-tilted” conformation of the receptor subunits (see Fig. 6B). In order to explain why S597 (site 2–1 peptide) is an agonist, whereas S961 (site 1–2 peptide) – agonist/antagonist, we suggest that S597 may not be capable of crosslinking the second pair partial sites and thus inactive the receptor as S961 does. We note that the distance between the actual receptor binding sites in these two peptides can be very different. If the receptor binding site in the site 1 peptide is positioned close to the N-terminus, and the receptor binding site of the site 2 peptide – close to the C-terminus, then a long distance between the receptor binding sites can be expected for the 1–2 peptide order (in the extended conformation of the peptide) as in S961, and a much shorter distance for the 2–1 peptide order as in S597 (see Fig. 6C). Thus, for the receptor binding sites positioned in S597 and S961 as in Fig. 6C, it is possible that the distance between the receptor binding sites in S961 is long enough for it to be capable of binding to the second crosslink and inactivate the receptor (Fig. 6C), but when the peptide order is reversed as in S597, the much shorter distance between the receptor binding sites (Fig. 6C) in S597 might prevent it from binding to the second crosslink. The proposed model is speculative, but consistent with the current knowledge of how insulin binds to the receptor [47]–[51]. Whether or not it is true requires further investigation and a better knowledge of the structure of the liganded receptor.
In summary, our results provide additional knowledge to the IR activation mechanism since we show that agonism and antagonism exist at IR. In addition, we provide in vitro studies which show that at 1 nM and 10 nM S961 can activate the IR and downstream signalling. Further exploration of the properties of such peptides should shed new light on the mechanism of IR activation and differential signalling.
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S661 antagonize IR and AKT phosphorylation in L6-hIR cells. Cells were incubated in 12-wells plates with a cell density of 125,000 cells/well for three days, where after the cells were stimulated with increasing concentrations of S661 (panel A and B) or HI (panelC and D) in the presence of 3 nM HI or 10 µM S661, respectively. IR (pY1158) tyrosine phosphorylation (panel A and C) as well as AKT (pS473) (panel B and D) was measured. Data points represent average of three experiments. Error bars show one standard deviation.
Figure S1.
S661 antagonize IR and AKT phosphorylation in L6-hIR cells. Cells were incubated in 12-wells plates with a cell density of 125,000 cells/well for three days, where after the cells were stimulated with increasing concentrations of S661 (panel A and B) or HI (panel C and D) in the presence of 3 nM HI or 10 µM S661, respectively. IR (pY1158) tyrosine phosphorylation (panelA and C) as well as AKT (pS473) (panel B and D) was measured. Data points represent average of three experiments. Error bars show one standard deviation. doi:10.1371/journal.pone.0051972.s001 (TIF)
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